Analysis of single molecules by EM provides a powerful approach to study the mechanics of DNA replication. The continuing focus in this renewal application is how eukaryotic origins are opened by Origin Recognition Complexes (ORC) and how DNA strands are looped at the fork to coordinate leading and lagging strand replication. A statistically large number of molecules are examined and this information combined, with data from biochemical assays. A highly interactive program has been established with Dr. Steve Bell at MIT (yeast ORC), Drs. Tom Broker and Louise Chow at UAB, (human papillomaviruses), Dr. Charles Richard at Harvard (T7 replication), and Dr. Nancy Nossal at the NIH (T 4 replication). Using several EM techniques, the structure of the yeast ORC will be examined alone, and bound to yeast ARS elements. A molecular pointer will localize individual subunits of the ORC, each individually tagged with biotin. EM will be used to explore the unwinding of the ARS by the ORC and other factors. The nature of eukaryotic origin recognition and opening will be further probed using the human papillomavirus. The ability of the viral E1 protein to unwind the origin in a reaction facilitated by E2 and host chaperone proteins will be examined. The role of host DNA polymerase alpha and cellular cyclin E and cdk2 kinase will be explored. The folding of the lagging strand at a moving T7 replication fork will continued to be probed using new mini- circle DNA templates. EM will be used to explore the question of now Okazaki fragment size is controlled and the role of newly discovered novel structural 'spools' created by the binding of single stranded DNA on the lagging strand by the T7 SSB. Using the T4 replication system studies will continue on the looping of the lagging strand in coordinating replication. The nature of the loop and proteins required will be examined. Each system offers a unique window into these questions and information garnered from one is immediately applied to the others. This grant has also provided a unique national resource for the development and application of EM methods for studying DNA and DNA-protein complexes and many NIH-funded investigators in need of help in this technology have been aided.